Dynamic stiffness and crossbridge action in muscle

Small sinusoidal vibrations at 300 Hz were applied to frog sartorius muscle to measure the dynamic stiffness (Young's modulus) throughout the course of tetanus. For a peak-to-peak amplitude of 0.4% the dynamic Young's modulus increased from 1.5×10⁵ Nm^–2 in the resting state to 2×10⁷ Nm^–2 in tetanus. After correction for the external connective tissue, the dynamic Young's modulus of the muscle was almost directly proportional to the tension throughout the development of tetanus. The ratio of dynamic Young's modulus to tensile stress thus remained constant (with a value at 300 Hz of approximately 100), consistently with Huxley and Simmons' identification of the crossbridges as the source of both tension and stiffness.For a single crossbridge the ratio of stiffness to tension was 8.2×10⁷ m^–1 at 300 Hz; it is deduced from literature data that the limiting value at high frequencies is about 1.6×10⁸ m^–1. This ratio is interpreted on Harrington's (1971) model to show that crossbridge action can be explained by a helix-coil transition of about 80 out of the 260 residues in each S-2 myosin strand. It is also shown that a helix-coil model can account for the observed rapid relaxation of muscle without invoking any complex behaviour of the crossbridge head.     

Studies in the mechanism of phenylethylamine uptake by rabbit erythrocytes

The mechanism of phenylethylamine (PEA) uptake by in vitro rabbit erythrocyte preparations involve both a larger component of passive diffusion and an Na+-dependent facilitated transport system. This is reflected by the fast rate and lack of saturation of PEA erythrocyte uptake, and by the decrease in PEA tissue/medium ratio when the amine was incubated in an Na+-free medium or in the presence of ouabain. Results obtained after the addition of iodoacetamide or by the use of glucose-free medium suggest the Na+-K+ gradient as the driving force responsible for operation of the carrier mechanism. Preliminary results indicate that amphetamine PEA share a similar mechanism for their uptake by rabbit erythrocytes. At the levels normally present in rabbit blood (c. 1 X 10(-8)M), about one-third of the PEA is found in the serum and only a very small portion of it (less than 1%) is present as the "biologically active" non-ionized amine. Most of the remaining phenylethylamine is bound to plasma protein or inside the erythrocytes.     

An electron spin resonance study of the novel radical cation produced during the horseradish peroxidase-catalyzed oxidation of tetramethylhydrazine

Thrombin stimulation of [³²P]-prelabeled platelets induces a rapid decrease of the radioactivity from phosphatidylinositol-4,5-bisphosphate. No significant change is observed in phosphatidylinositol-4-monophosphate. The initial, thrombin-induced decrease of phosphatidylinositol-4,5-bisphosphate is not inhibited by cytochalasin D or by compounds that interfere with the mobilization of Ca²⁺ such as 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the calmodulin-antagonist, trifluoperazine, prostacyclin and cyclic AMP. Our information indicates that the rapid loss of phosphatidylinositol-4,5-bisphosphate is linked to receptor activation and insensitive to Ca²⁺-mobilization.     

The IgM-associated protein mb-1 as a marker of normal and neoplastic B cells.

Recent evidence indicates that the transmembrane form of IgM on murine and human B lymphocytes is physically associated with at least two proteins, forming a disulfide-linked dimer, which may control cell surface expression of IgM and also play a role in signal transduction after Ag binding (by analogy with the TCR-associated CD3 components in T lymphocytes). We have used mAb and polyclonal antibodies against an intracytoplasmic epitope on one of these polypeptides (previously identified in murine B cells as the product of the B cell specific mb-1 gene) to study the distribution of the IgM-associated dimer in human cells. By immunocytochemical staining of normal and neoplastic B cells, we show that the human mb-1 protein appears early in B cell differentiation, probably before expression of cytoplasmic mu-chain, and persists until the plasma cell stage, where it is seen as an intracytoplasmic component. According to immunohistologic analysis of reactive lymphoid tissue and lymphoma samples, mb-1 protein is completely B cell specific. Anti-mb-1 also labels B cell areas in tissues from seven different mammalian species. Finally, the Ig-associated dimer could be isolated from human hairy-cell leukemia cells in high purity and yield by affinity chromatography using anti-mb-1 antibody. Mice immunized with this material have produced a strong polyclonal response, so that it should now be possible to prepare a panel of new mAb reactive with different epitopes on both mb-1 and on its associated polypeptide(s).     

The membrane IgM-associated heterodimer on human B cells is a newly defined B cell antigen that contains the protein product of the mb-1 gene

7/8embrane IgM (mIgM) on human B lymphocytes is noncovalently associated with a disulfide-linked dimer that contains phosphoproteins of 47 and 37 kDa. In this study, the biochemical properties and the identity of these Ag receptor-associated components have been addressed. Both subunits carry N-linked carbohydrate groups. After deglycosylation, the 47-kDa and 37-kDa proteins have similar molecular masses, of about 23 kDa, and relatively acidic but different isoelectric points. The accumulated data, together with a previously performed comparison of tryptic peptides, suggest that the two components are structurally distinct and possibly encoded by different genes. Indeed, a mAb, raised against a synthetic peptide that was made on the basis of the published carboxyl-terminal amino acid sequence of the human mb-1 gene product, specifically reacted with the 47-kDa but not the 37-kDa subunit. None of the established B cell-specific mAb characterized in the Fourth International Workshop on Leukocyte Antigens, including CD24, CD37, and CD72, detect the mIgM-linked heterodimer, which makes it a newly defined human B cell Ag.     

Prevalence and isolation of Toxoplasma gondii from white-tailed deer in Alabama

Nineteen white-tailed deer (Odocoileus virginianus) from 5 counties in Alabama were examined for infection with Toxoplasma gondii. Twenty-gram samples of heart tissue were bioassayed in mice, serum was examined for T. gondii antibodies using the direct agglutination test, and sections of heart muscle were examined histologically for tissue cysts. Toxoplasma gondii was isolated from 4 of 19 (21%) white-tailed deer hearts. Antibody titers of greater than or equal to 1:50 were found in sera from 7 of 16 (44%) white-tailed deer. Histological examinations of tissue sections from white-tailed deer hearts were negative for T. gondii.     

Rat procathepsin B. Proteolytic processing to the mature form in vitro.

Expression of rat procathepsin B in yeast led to the secretion of both the latent and mature forms of the enzyme. Culture in the presence of a cysteine proteinase inhibitor prevented this processing. We have expressed and purified a mutant form of rat procathepsin B whose active-site cysteine residue has been changed to a serine, and which also lacks the glycosylation site in the mature region of the protein. This non-active mutant protein was secreted essentially in an unprocessed form. The purified protein has been incubated with a variety of proteinases, and results indicate that cathepsins D and L, as well as mature cathepsin B itself, can produce a processed (single-chain) form of cathepsin B from this precursor. Amino-terminal sequencing of these processed forms has revealed that they are all elongated by a few residues with respect to the mature form found in vivo. The action of a combination of cathepsin B with dipeptidylpeptidase I produced a single-chain form of cathepsin B with the correct amino terminus. This work has also shown that the processing of procathepsin B to a single-chain form can be an autocatalytic process, in at least an intermolecular manner.     

Drug-Induced Changes in Blood Pressure Lead to Changes in Extracellular Concentrations of Epinephrine, Dihydroxyphenylacetic Acid, and 5-Hydroxyindoleacetic Acid in the Rostral Ventrolateral Medulla of the Rat

Neurochemical changes in the extracellular fluid of the rostral ventrolateral medulla (RVLM) were produced by changes in arterial blood pressure. Blood pressure was raised or lowered with systemic infusions of phenylephrine or nitroprusside and neurochemicals were recovered from RVLM by in vivo microdialysis. A dialysis probe 300 microns in diameter and 500 microns in length was stereotaxically implanted in the RVLM of the urethane-anesthetized rat. Sterile physiological Ringer's solution was perfused at a rate of 1.5 microliter/min. The perfusate was collected under ice-cold conditions every 15 min for the assay of epinephrine, dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), ascorbic acid, and uric acid. After stable baseline neurochemical concentrations were achieved, animals were infused with phenylephrine or nitroprusside intravenously to raise or lower the blood pressure. Increasing blood pressure 50 mm Hg above the baseline value by phenylephrine led to a significant reduction in heart rate and a reduction in extracellular epinephrine and DOPAC concentrations. The 5-HIAA concentration was increased during the hypertensive drug infusion. There were no changes in the concentrations of ascorbic acid or uric acid. Hypotension produced by nitroprusside (-20 mm Hg) led to neurochemical changes which were the reciprocal of those seen during hypertension. During hypotension, heart rate increased as did the extracellular fluid epinephrine concentration. The 5-HIAA concentration fell with hypotension and remained depressed following the nitroprusside infusion. Ascorbic acid and uric acid concentrations did not change during hypotension but ascorbic acid did increase after the nitroprusside infusion stopped. These data provide direct evidence that epinephrine release in RVLM is linked to changes in systemic blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence of a cDNA encoding porcine testis 17 alpha-hydroxylase cytochrome P-450.

We describe the isolation and characterization of a cDNA encoding the complete porcine neonatal testis 17 alpha-hydroxylase/C-17,20-lyase cytochrome P-450. The deduced amino acid sequence is 509 amino acids in length.